TRIPLE E PROBES ACTIVITY-BASED PROBES FOR E1-E2

TRIPLE E PROBES
ACTIVITY-BASED PROBES FOR E1-E2-E3 ENZYMES
With UbiQ’s revolutionary activity-based E1-E2-E3 probe it has become possible to crystallize, identify
or validate dozens of enzymes, or potential targets, involved in protein ubiquitination. The probe is
processed in a native way by the E1-E2-E3 cascade. In addition to this it has the option of reacting with the
active site cysteine residues in an irreversible way, thus trapping the enzymes in the ubiquitination cascade.
3. Triple E probe shuttles through ubiquitination cascade...
Ub
O
Ub
O
ATP
OH
Ub
AMP
O
S
E1
1. Inactive
Triple E probe
2. Triple E probe
activated by E1
O
S
Ub
O
Ub
S
E2
E1
O
S
E3
HO
O
HO
O
HO
O
Ub
S
Ub
S
Ub
S
E1
E2
E3
4. ...or irreversibly traps active enzymes in the cascade.
figure 1
Triple E probes contain a C-terminal dehydroalanine (Dha) group, which at physiological pH is relatively
inert on its own but becomes very electrophilic upon activation by the E1 enzyme (figure 1). While the
probe is processed in a native way by the E1-E2-E3 cascade, the Dha group can react with the active site
cysteine residues in an irreversible way, thereby trapping the E1, E2 and E3 enzymes (figure 1).
In contrast to other probes 2, our Triple E probes represent the first tools that allow the monitoring of full
E1-E2-E3 cascade activity.1
applications
• gel based activity protein profiling, see appendix figure 2
• cell based assays, see appendix figure 3
• structural biology, see appendix figure 4
1
(a) Mulder et al., Nat. Chem. Biol. 2016, 2084 [Epub ahead of print]
(b) MPC Mulder, F. El Oualid and H. Ovaa. Adenylation enzyme inhibitors. Patent: PCT/NL2015/050596 (publication date 03 March 2016).
2(a) Lu et al. J. Am. Chem. Soc. 2010, 132, 1748.
(b) Olsen et al. Nature, 2010, 463, 906.
(c) H. An and A.V. Statsyuk, J. Am. Chem. Soc. 2013, 135, 16948.
(d) H. An and A.V. Statsyuk Chem. Comm. 2016, 52, 2477.
(e) Kuan-Chuan Pao et al. Nat. Chem. Biol. 2016, 2045 [Epub ahead of print]
3
Page et al. Biochemistry 2012. 51, 4175.
Science Park 408, 1098 XH Amsterdam, The Netherlands
[email protected]
+31 20 303 1970
www.ubiqbio.com
fact sheet, May 2016
reference
TRIPLE E PROBES - ACTIVITY-BASED PROBES FOR E1, E2, E3 ENZYMES
CATALOGUE OF TRIPLE E PROBES
UbiQ offers a selection of Triple E probes with various substrates and N-Terminal tags. For custom development
please contact UbiQ directly.
Tag
Substrate
Electrophile Code
Name
−
Ub
Dha
UbiQ- 101
Ub-Dha
Biotin
Ub
Dha
UbiQ-102
Biotin-Ahx-Ub-Dha
His6
Ub
Dha
UbiQ-103
His6-Ahx-Ahx-Ub-Dha
Cy5
Ub
Dha
UbiQ-104
Cy5-Ub-Dha
5-carboxyRh110
Ub
Dha
UbiQ-131
5-carboxyRh110-Ub-Dha
−
Nedd8
Dha
UbiQ-105
Nedd8-Dha
Biotin
Nedd8
Dha
UbiQ-106
Biotin-Ahx-Nedd8-Dha
5-carboxyRh110
Nedd8
Dha
UbiQ-122
5-carboxyRh110-Nedd8-Dha
Triple E probes
Tailor made
A series of proprietary techniques give us structural control on all aspects of our reagents, enabling us to construct
reagents that are beyond the reach of any currently available alternative approaches.
Please contact us for more information
FAQ
Which of the E1-E2-E3 enzymes the probe will target?
The probe targets active site cysteine based E1-E2-E3 enzymes.1
What about RING class E3 enzymes that lack a catalytic site cysteine and thus are not directly targeted
by the Triple E probe?
Highly stable E2-Ub(l)-Dha thioether adducts act as stable (competitive) inhibitors of RING type E3 ligases.
This allows studies of the RING class E3 enzymes.1
Is the probe transferred to substrates of the E1-E2-E3 enzymes?
As far as we know the Triple E Probe is not transferred to substrates.1a
How specific is the probe labelling?
The ubiquitin (-like) substrate context of the probe in combination with the required activation by the E1
mediated adenylation step, makes the probe highly specific for enzymes in the ubiquitination cascade.1
Can the probe be used in cell lysates and live cells?
Yes, see Figure 2 and Figure 3a and 3b.1
fact sheet, May 2016
Is the probe processed in a native way?
Yes, see figure 1.1a
Are other substrates or tags available for the probe?
Yes, other substrates will follow soon, or can already be tailor-made upon request.1
Science Park 408, 1098 XH Amsterdam, The Netherlands
[email protected]
+31 20 303 1970
www.ubiqbio.com
TRIPLE E PROBES - ACTIVITY-BASED PROBES FOR E1, E2, E3 ENZYMES
APPENDIX
The Triple E probes allow the monitoring of full E1-E2-E3 cascade activity. The probes have been tested in various applications.
Please see reference 1 for the details on the experimental set-up.
1 ACTIVITY-BASED PROTEIN PROFILING IN CELL EXTRACT
figure 2 – In-gel fluorescence imaging of
A) a mixture of E1 (Uba1), E2 (Ube2L3) and E3 (ITCH) and
B) a panel of tumor cell lines, all incubated with Cy5-Triple E
probe and ATP.
2 CELL-BASED ASSAYS
electroporation
fixation &
imaging
A
GFP-UBE2J1
B
GFP-C91S
C
PYR-41
GFP-UBE2J1
overlay
untransfected
Cy5-Ub-Dha
zoom
GFP-UBE1
GFP
overlay
DAPI
Cy5-Ub-Dha
scale bars = 10 µm
figure 3b – visualizing E2 activity in HeLa cells
with (electroporated) Cy5-Ub-Dha (magenta).
GFP-UbE2J1 = green, green + magenta = white.
PYR-41= Uba1 inhibitor.1a
fact sheet, May 2016
figure 3a – distribution of Cy5-Ub-Dha (magenta) in HeLa cells
that electro ectopically express GFP-UBE1 (green) relative to
untransfected cells.1a
Science Park 408, 1098 XH Amsterdam, The Netherlands
[email protected]
+31 20 303 1970
www.ubiqbio.com
TRIPLE E PROBES - ACTIVITY-BASED PROBES FOR E1, E2, E3 ENZYMES
APPENDIX
3 STRUCTURAL BIOLOGY
UBE2D3
His75
UBE2D3
Asp87
Ub
O
UBE2D3
Arg90
S
E2
UBE2D3
Asn77
UBE2D3
Asp117
figure 4 – Structural analysis [Ub-Dha-UBE2D3] conjugate (formed via Ub E1). (1, 3)1a
4 PROTEOMICS
fact sheet, May 2016
figure 5 – proteome-wide activity profiling of Ub ligation in HeLa cells.1
Science Park 408, 1098 XH Amsterdam, The Netherlands
[email protected]
+31 20 303 1970
www.ubiqbio.com